The TIRF microscope technique is a high SN-ratio observation method capable of local excitation on nano-scales. This technique has been widely used for observation of cell membrane activities and single-molecule events in the cell biological field (see Non-Patent Publication 1), and it has made much contributions to experimental revelation of the electrical properties (Non-Patent Publication 1) or Brownian movement of colloidal particles in the electrochemical field as well (Kihm, K. D. et al., Exp. in Fluids, 37, pp. 811-824, (2004). The most noticeable feature of this method is that for fluorescent observation, there are evanescent waves used as an excitation light source, which are generated in association with total internal reflection at an interface of two substances having different refractive indexes. FIG. 1 is illustrative of how the evanescent waves are generated; when, on the interface of a substance 1 having a refractive index n1 and a substance 2 having a refractive index n2, light is incident from the side of the substance 2 having a higher refractive index at an angle larger than the critical angle, the light is subjected to total internal reflection at that interface. However, there are some evanescent waves unavoidably generated from the interface to the side of the substance 1 having a lower refractive index, which waves attenuate exponentially. The evanescent waves are light showing up slightly from the total internal reflection interface to an area of about a few tens to a few hundred nm. In the TIRF technique, therefore, evanescent waves are generated at an interface of a sample stained with fluorescent dye and a slide or cover glass (hereinafter called the slide glass) to enable high SN-ratio fluorescent observation at only a limited sample site.
What is now commercially available and generally often used is mainly such an objective lens type TIRF microscope as shown in FIG. 14 (a). More specifically, an objective lens of the inverted type is positioned below the slide glass via an oil-immersion oil, and evanescent wave-generation laser light is obliquely incident from below the slide glass via that objective lens so that evanescent waves are generated near the interface with a sample placed on the slide glass. This arrangement performs well and is convenient because the space above the objective lens is freely accessible, and it gives a very bright fluorescent image as well. However, the principal requirement for use of a high-aperture, oil immersion objective lens is supposed to limit observation to one at magnifications of as high as 60 or greater.
A prism type TIRF microscope adapted to enter laser via a prism such as the one shown in FIG. 14(b) has been widely used, too. In this case, a sample is held between two slide glasses with the prism placed on one side glass, and evanescent wave-generation laser light is entered obliquely up in the upper slide glass to generate evanescent waves near the interface of the slide glass in contact with the sample. This arrangement is capable of high SN-ratio observation because of efficient incidence of laser light, and enables low-magnification observation to be easily implemented because of no restriction on magnification at all. However, the space above the objective lens is closed up, resulting in very poor specimen manipulation and more limited degrees of sample flexibility.
As described above, the TIRF microscopes are still far away from meeting the need of making simultaneous comparisons at a plurality of samples of reactions that cells exhibit to various chemicals applied in the process of experimentation. Thus, there is a mounting demand for the development of a TIRF microscope that makes sure good enough specimen manipulation and sample flexibility, facilitates combined use with other optical observation methods, and enables observation at low magnifications.
Past studies include Non-Patent Publication (3) wherein, as shown in FIG. 15(a), an entrance prism and a radiation prism are bonded to the underside of a slide glass; laser light is introduced from the entrance prism into the slide glass where it is subjected to multiple total internal reflection; during that multiple total internal reflection, evanescent waves are generated near the upper surface of the slide glass to excite a specimen; and the laser light guided by multiple total internal reflection goes out via the radiation prism, and Non-Patent Publication 4 wherein, as shown in FIG. 15(b), an end face of a slide glass is processed into an inclined one; laser light is introduced from that inclined end face into the slide glass to subject it to multiple total internal reflection; and upon that multiple total internal reflection, evanescent waves are generated near the upper surface of the slide glass to excite a specimen; and the laser light guided by multiple total internal reflection goes out from the opposite end face of the slide glass. These methods take hold of a free space above the sample and facilitates low-magnification observation, but because the slide glass is limited to a small thickness (0.17 mm in the former, and 0.2 mm in the latter), they offer some problems: a lot more multiple total internal reflections, the occurrence of scattered light due to total internal reflection, attenuation of guided light, the likeliness of the specimen to discolor due to fluorescence, and lower SN ratios. Further, the positions of incidence and radiation of laser light remain fixed, rendering it hard to adjust an optical path for laser light, and making sample manipulation not easy because of the need of moving the objective lens to change a specimen observation position. Yet further, much work is needed with decreased versatility, because of the need of bonding the prisms to the slide glass for each sample.
Non-Patent Publication 1                Axelrod, D., Traffic, Vol. 2, pp. 764-774, (2001)        
Non-Patent Publication 2                Prieve, D. C, and Frej. N. A., Langmuir, 6, pp. 396-403, (1990)        
Non-Patent Publication 3                Conibear, P. B. and Bagshaw, C. R., Journal of Microscopy, Vol. 200, Pt 3, pp. 218-229, (2000)        
Non-Patent Publication 4                Teruel, M. N, and Meyer, T., Science, Vol. 295, pp. 1910-1912, (2002)        